Labeling of Denatured Collagen in ADPKD and Comparison to Fibrotic Area

Background: Autosomal dominant polycystic kidney disease (ADPKD) is the fourth most common cause of kidney failure, and manifests through the proliferation of countless kidney cysts. Mounting research suggests the extracellular matrix (ECM) is integral to disease progression. Collagen, the predominant protein within the ECM, is encoded by 44 genes, forming intricate supramolecular structures that constitute 28 distinct collagen types. Methods used in ADPKD for tracking total collagen histologically are primarily trichrome or Picrosirius red (PSR) staining to determine percent fibrotic area. Besides fibrosis, destruction of collagen assemblies is a hallmark of tissue injury and disease. We examined here if denatured collagen is a more sensitive disease indicator than fibrotic area. Methods: Pkd2hi4166 zebrafish were used with heterozygous adults compared to sibling WT. Pkd1RC/RC mice were used in C57BL/6J and compared to wildtype (Jackson Laboratory). Collagen hybridizing peptide (CHP, 3Helix) was used for fluorescence labeling of denatured collagen. CHP labeling was compared to fibrotic area determined from PSR staining. PSR staining, CHP labeling, and tissue area were calculated using ImageJ. Data are shown as mean ± SEM, p-values by t-test or ANOVA. Results: CHP labeling surrounded 47.9% ± 14.5 of tubules in pkd2+/- zebrafish (n=8 fish), while no labeling was seen in WT (n=10; p=0.002). Labeling occurred around both proximal tubules/collecting ducts (LTL+; 41.7% ± 25.0, n=4) and distal tubules (DBA+; 54.0% ± 18.2, n=4). Labeling in mouse was not clearly associated with individual tubules and was present in normal and cystic regions. Quantification of fluorescence-tagged CHP in entire kidney sections showed more labeling in Pkd1RC/RC mice (581 au ± 68.3; n=6 mice) vs WT (341 au ± 14.4; n=6; p=0.006). Conclusion: Denatured collagen labeling by CHP showed more consistent differences than fibrotic area by PSR. Additionally denatured collagen was detected distant from cysts in Pkd1RC/RC mice and in pkd2+/- zebrafish which lack cysts. These data suggest measuring denatured collagen may allow more reliable and sensitive detection of collagen changes in ADPKD than fibrotic area. Funding: NIDDK Support